Efficient expression and characterization of a cold-active endo-1, 4-β-glucanase from Citrobacter farmeri   by co-expression of Myxococcus xanthus   protein S

Background: Cold-active endo-1, 4-β-glucanase (EglC) can decrease energy costs and prevent product denaturation in biotechnological processes. However, the nature EglC from C. farmeri A1 showed very low activity (800 U/L). In an attempt to increase its expression level, C. farmeri EglC was expressed in Escherichia coli

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as an N-terminal fusion to protein S (ProS) from Myxococcus xanthus.
Results: A novel expression vector, pET(ProS-EglC), was successfully constructed for the expression of C. farmeri EglC in E. coli. SDS-PAGE showed that the recombinant protein (ProS-EglC) was approximately 60 kDa. The activity of ProS-EglC was 12,400 U/L, which was considerably higher than that of the nature EglC (800 U/L). ProS-EglC was active at pH 6.5–pH 8.0, with optimum activity at pH 7.0. The recombinant protein was stable at pH 3.5–pH 6.5 for 30 min. The optimal temperature for activity of ProS-EglC was 30°C–40°C. It showed greater than 50% of maximum activity even at 5°C, indicating that the ProS-EglC is a cold-active enzyme. Its activity was increased by Co²⁺ and Fe²⁺, but decreased by Cd²⁺, Zn²⁺, Li⁺, methanol, Triton-X-100, acetonitrile, Tween 80, and SDS.
Conclusions: The ProS-EglC is promising in application of various biotechnological processes because of its cold-active characterizations. This study also suggests a useful strategy for the expression of foreign proteins in E. coli using a ProS tag.

Keywords
Cellulose degradation; Cellulose; Cold-active enzyme; Endoglucanases; Enzymatic properties; Escherichia coli; Expression; Novel expression vector; N-terminal fusion; Protein S-tag; Recombinant protein